Viral nucleotide sequences

ABSTRACT

Various genes of herpes virus of turkeys (HVT), Marek&#39;s disease virus (MDV) and infectious laryngotracheitis virus (ILTV) have been identified as non-essential regions (and candidates for insertion sites for foreign genes) and/or as antigen-encoding regions. The former include the HVT homologue of the HSV (herpes simplex virus) gC gene, the TK (thymidine kinase) region of MDV or ILTV, ORF3 of ILTV (as defined herein), the ribonucleotide reductase (large subunit) gene of ILTV, MDV or HVT and the ribonucleotide reductase (small subunit) gene of MDV. The antigen-encoding regions include the HVT homologues of the HSV gB, gC and gH genes, the ILTV homologue of HSV gB, ORF2 of ILTV, and the HVT homologue of the HSV-1 immediate early genes IE-175 and IE-68.  
     Manipulation of these genes allows vaccines to be prepared comprising attenuated virus or virus carrying heterologous antigen-encoding sequences.

[0001] The present invention relates to viral nucleotide sequences whichmay be manipulated to provide vaccines against disease.

BACKGROUND AND DESCRIPTION OF PRIOR ART

[0002] Herpesviruses are large double stranded DNA viruses consisting ofan icosahedral capsid surrounded by an envelope. The group has beenclassified as alpha, beta and gammaherpesviruses on the basis of genomestructure and biological properties [Roizman, B et al (1981)Inter-virology 16, 201-217]. Avian herpes viruses include Marek'sDisease Virus (MDV) (a gammaherpesvirus) which causes a lymphomatousdisease of considerable economic importance in chickens [reviewed inPayne, L. N. (ed) Marek's Disease (1985), Martinus Nijhoff Publishing,Boston] and Infectious Laryngotracheitis Virus (ILTV) (analphaherpesvirus) which causes an acute upper respiratory tractinfection in chickens resulting in mortality and loss of egg production.

[0003] A recent unexpected finding in out laboratory is that there issufficient amino acid homology between MDV, ILTV and mammalianherpesviruses, particularly varicella zoster (VZV) and Herpes Simplexvirus (HSV) to allow identification of numerous conserved genes. Theseinclude the MDV and Herpesvirus of Turkeys (HVT) homologues ofglycoproteins gB, gC and gH of HSV; the ILTV, MDV and HVT homologues ofTK and ribonucleotide reductase genes and the ILTV homologue of COB andgenes 34 and 35 of VZV [Buckmaster, A et al, (1988) J. gen. Virol, 69,2033-2042.

[0004] Strains of MDV have been classified into three serotypes. Type 1comprises pathogenic strains and their attenuated derivatives. Type 2are a group of naturally-occurring non-pathogenic strains and type 3 isHVT. For more than a decade, vaccination with HVT has been remarkablyeffective in controlling Marek's disease. However, in recent years, newstrains of MDV have been isolated which cause disease despitevaccination with HVT. Losses due to these ‘very virulent’ strains haveoccurred in parts of the U.S.A., Europe and the Middle East. Althoughthe degree of protection can be improved by using a mixture of HVT, type2 MDV and attenuated derivatives of very virulent strains forvaccination, the results have been erratic. These observations and thefact that there are MDV type-specific epitopes that are not shared byHVT or type 2 MDV have led us to the conclusion that improved vaccinesmight be constructed which are antigenically more related to MDV thanexisting vaccines. [Reviewed by Ross and Biggs in Goldman J. M. andEpstein M. A. (eds) Leukaemia and Lymphoma Research, VaccineIntervention against Virus-Induced Tumour, p 13-31, Macmillan, 1986].

[0005] Infectious laryngotracheitis is also a worldwide problem.Sporadic outbreaks occur in which the severity of clinical symptomsvaries considerably. Virus can persist in birds that have recovered andmay be shed at intermittent intervals after recovery. An attenuatedfield strain is currently used as a vaccine. However, it has retainedsome degree of pathogenicity. Mortality due to the vaccine may reach 10%in young chicks.

[0006] A number of herpesvirus antigens have been shown to conferprotective immunity when expressed in a recombinant vaccinia virus.These include the gB gene of HSV [Cantin E. M. et al (1987) Proc. Natl.Acad. Sci. U.S.A. 84, 5908-5912], gD of HSV [Paoletti, E. et al (1984)Proc. Natl. Acad. Sci. U.S.A. 81, 193-197] and gp50 of pseudorabiesvirus (PRV), a homologue of HSV gD [Marchioli, C. C. et al (1987) J.Virol. 61, 3977-3981]. Because of the absolute requirement of gB forvirus penetration and infectivity and because it is conserved amongherpes-viruses, gB and its homologues are important immunogens.Moreover, the presence of gB at the surface of infected cells has beenshown to be an important target for humoral and cell-mediated immuneresponses [Blacklaws, B. A. et al J.gen. Virol. 68, 1103-1114 (1987);McLaughlin-Taylor, E. et al (1988) J. gen. Virol. 69, 1731-1734]. Therecently described glycoprotein gH of HSV is also essential forinfectivity and may also be an important immunogen (Desai, P. J. et al(1988) J. gen. Virol. 69, 1147-1156]. It has also been shown that gIIIof pseudorabies virus (PRV), a homologue of gC, is a major target forneutralizing antibody and for cytotoxic T cells although it is anon-essential protein. Also of interest is the unexpected participationof immediate early proteins in T cell mediated cytotoxic reactions incells infected with cytomegalovirus (CMV) [Kozinowski U. H. et al (1987)J. Virol. 61, 2054-2058]. Similar antigens could play an important rolein the rejection of latently infected and transformed lymphocytes inMarek's disease since immediate early RNA transcripts have been detectedin lymphoblastoid cell lines established from Marek's disease tumours.

[0007] Although many recombinant vaccines have been constructed usingthe poxvirus vaccinia as a vector, there are also reports of the use ofherpesviruses as vectors for the expression of foreign genes. Thushepatitis antigen has been expressed in HSV [Shih, M. F. et al (1984)Proc. Natl. Acad. Sci. U.S.A. 81, 5867-5870] and human tissueplasminogen activator has been expressed in PRV [Thomsen, D. R. et al(1987) Gene 57, 2-61-265. In both cases, foreign genes were inserted incloned fragments of non-essential herpes genes which were thenintroduced into the virus vector by homologous recombination. Thehepatitis virus gene was fused to a herpesvirus promoter and therecombinant DNA was inserted within the TK gene of HSV. Homologousrecombination following co-transfection of the recombinant DNA andwild-type HSV DNA resulted in TK⁻ virus clones that expressed thehepatitis antigen.

[0008] In the case of PRV, the gX gene mapping in U_(s) was used as thesite for insertion of the foreign gene. The strategy used involvedinsertion of the TK gene of HSV in the gX gene of a PRV mutant that hada defect in its TK gene resulting in a TK positive virus. The humantissue plasminogen activator gene was then inserted within a clonedfragement of HSV TK and the recombinant was introduced into the PRVmutant by homologous recombination. TK⁻virus was selected whichexpressed the human gene (Thomsen et al as above). Similarly, VZV hasbeen used as a vector [Lowe et al (1987) Proc. Natl. Acad. Sci. U.S.A.84, 3896-3900].

[0009] Several herpesvirus genes have also been shown to be associatedwith virulence and to be non-essential for growth in vitro. Theseinclude the TK genes of HSV [Jamieson, A. T. et al (1974) J. gen. Virol.24, 465-480; Field, H. and Wildy, P., (1987) J. Hygiene (Cambridge) 81,267-277] and of PRV. Indeed it has long been known that PRV is readilyattenuated by deletion of TK activity [Tatarov, G. (1968) Zentralbl.Vet. Med 15B, 848-853]. Furthermore, attenuation of the Bartha strain ofPRV has been attributed to a defect in gI, a non-essential structuralglycoprotein mapping in U_(s) [Mettenleiter, T. et al (1987) J. Virol.61, 4030-4032].

[0010] Genes of HSV mapping in the internal repeat region (TRS) flankingthe long unique sequence have also been associated with pathogenicity[Rosen, A. et al (1986) Virus Research 5, 157-175; Thompson, R. L. et al(1983) Virology 131, 180-192]. Several additional genes of HSV have beenshown to be non-essential for growth in vitro although it is not knownwhether they are associated with virulence. These include UL24 (Sanders,P. G., (1982), J. gen. Virol. 63, 277-295, large subunit ofribonucleotide reductase (Goldstein D. J. and Weller, S. K. (1988) J.Virol. 62, 196-205), gC (Draper K. G. et al (1984) J. Virol. 51,578-585), dUTPase (Fisher, F. B. & Preston, V. G. (1986) Virology 148,190-197), and U_(L)55 and U_(L)56 (MacLean, A. R. & Brown, S. M. (1987)J. gen. Virol. 68, 1339-1350).

[0011] Moreover there is evidence that several genes of HSV mapping inU_(s) are also non-essential for growth in vitro [Weber, P. C. et al(1987) Science 236, 576-579].

SUMMARY OF THE INVENTION

[0012] One aspect of the present invention provides a nucleotidesequence substantially free of the sequences which would adjoin it inthe wild-type virus associated with the sequence, the sequence beingselected from the group consisting of:

[0013] (a) the HVT homologue of the HSV gB gene,

[0014] (b) the HVT homologue of the HSV gC gene,

[0015] (c) the HVT homologue of the HSV gH gene,

[0016] (d) the TK gene of ILTV,

[0017] (e) the ILTV homologue of the HSV gB gene,

[0018] (f) ORF2 of ILTV,

[0019] (g) ORF3 of ILTV,

[0020] (h) the ribonucleotide reductase (large subunit) gene of ILTV,

[0021] (i) the ribonucleotide reductase (large subunit) gene of HVT,

[0022] (j) the ribonucleotide reductase (large subunit) gene of MDV,

[0023] (k) the ribonucleotide reductase (large subunit) gene of MDV,

[0024] (l) the HVT homologue of the immediate early gene IE-175 ofHSV-I, and

[0025] (m) the HVT homologue of the immediate early gene IE-68 of HSV-I,

[0026] and minor variations thereof.

[0027] Each of sequences (a) to (m) may be associated with furtherelements such as suitable stop and start signals and other 5′ and 3′non-coding sequences, including promoters, enabling expression of thesequence. Such further elements may be those associated with thesequence in its naturally-occurring state or may be heterologous to thatsequence.

[0028] In particular the promoter may be one associated with one of thesequences (l) and (m) above.

[0029] The term “minor variations thereof” is intended to includechanges in the nucleotide sequence which do not affect its essentialnature, for example minor substitutions of nucleo-tides for one another.In the case of sequences which are intended for insertion into a vectorto encode an antigen, the “essential nature” of the sequence refers tothe (glyco)protein encoded. Conservative changes in the nucleotidesequence which give rise to the same antigen will clearly be included,as will changes which cause conservative alterations in the amino acidsequence which do not affect adversely the antigenic nature of theantigen, in particular, antigenic portions of the antigen sequences maybe used alone, for example the regions corresponding to nucleotides273-320 or 867-926 of HVT gH and minor variations thereof. Thesesequences and the peptides encoded thereby form a further aspect of theinvention. In the case of a sequence which is an insertion site, it isnecessary only that the sequence should be non-essential for theinfectivity and replication of the virus and have sufficient homologywith the defined sequence to enable recombination to occur. Thus aninsertion of one nucleotide into the sequence could completely changethe reading frame from then on in a downstream direction. In the case ofan antigen-encoding sequence this would usually alter the amino acidsequence undesirably (depending on where the frameshift occurred), butin the case of an insertion site, the degree of homology would be almostthe same, thereby allowing recombination to take place with almost thesame ease.

[0030] Generally speaking, in an insertion site, if a nucleotidehomology of at least 75% is present, the sequence is regarded as a“minor variation”. Preferably, the sequence is at least 80, 85, 90, 95or 99% homologous.

[0031] It will be appreciated that such degrees of homology relate tosubstantially the entire portion of each sequence (a) to (m) definedabove. Shorter sequences may be used as probes in the identification orisolation of such longer sequences, but in this case the degree ofhomology will in general need to be greater in order to ensure accuratehybridisation.

[0032] Thus, a further aspect of the invention provides sub-sequences ofat least 13 nucleotides having at least 90% (preferably 95%, 99% or100%) homology with at least one portion of any of the said sequences(a) to (m) above.

[0033] In the above list, sequences (a) to (c), (e), (f), (l) and (m)are useful for expressing viral antigens. Sequences (b), (d) and (g) to(k) and, in addition, the TK region of MDV are useful as non-essentialsites suitable for insertion of antigen-expressing genes. Thus, sequence(b) is useful for both functions.

[0034] The sequences may readily be isolated from naturally-occurringILTV, HVT and MDV viruses, using the sequence information given hereinand standard techniques, for example involving the preparation ofoligonucleotide probles and use thereof to hybridise to thenaturally-occurring DNA.

[0035] Antigenic ILTV and HVT sequences, i.e. sequences (a) to (c), (e),(f), (l) and (m) above, may be expressed in any suitable host and, inparticular, in HVT or MDV. Suitable non-essential sites for insertion ofone ILTV sequence include the MDV homologue of the HSV gC gene, the HVThomologue of the HSV gC gene, the TK gene of HVT or MDV, theribonucleotide reductase (large subunit) gene of HVT or MDV and theribonucleotide reductase (small subunit) gene of MDV.

[0036] A second aspect of the invention provides insertional ordeletional mutants of MDV, HVT and ILTV as follows:

[0037] (i) for HVT, a mutation in the region homologous to the HSV gCgene or in the ribonucleotide reductase gene or the TK gene,

[0038] (ii) for MDV, a mutation in the region homologous to the HSV gCgene or in the ribonucleotide reductase (small subunit) gene or in theribonucleotide reductase (large subunit) gene,

[0039] (iii) for ILTV, a mutation in the TK gene, ORF3 or theribonucleotide reductase (large subunit) gene.

[0040] Each mutation may be in the coding or non-coding sequences of theregions identified.

[0041] Such mutant forms of HVT. MDV and ILTV may be used as, or createdin the course of preparing, viral vectors for heterologousantigen-encoding sequences, or indeed as vectors for any other sequencewhich one wishes to express in a fowl in which the vector willreplicate. Such sequences include, but are not limited to, (a) to (c),(e), (f), (l) and (m).

[0042] By “heterologous”, we mean that the antigen-expressing sequencehas not previously been found in the same place in relation to theremainder of the viral genome. For example, an antigen-expressing genemight be isolated from a virulent strain of ILTV and inserted into theTK region of a less virulent strain of ILTV; this insertion would beregarded as “heterologous” if it did not result in a naturally-occurringvirus.

[0043] The heterologous sequence may alternatively be one coding for anantigen associated with any one of the following diseases: avianencephalomyelitis (epidemic tremor), avian influenza (fowl plague),avian leukosis, avian paramyxoviruses other than Newcastle disease (PMV2to PMV7), avian reovirus diseases (enteric disease, tenosynovitis),chicken anaemia (caused by chicken anaemia agent), coccidiosis, egg dropsyndrome (EDS76), fowl pox, infectious bronchitis, infectious bursaldisease (Gumboro), inclusion body hepatitis (adenovirus),lymphoproliferative disease of turkeys, Newcastle disease,reticuloendotheliosis in chickens, reticuloendotheliosis in turkeys,rotavirus enteritis, turkey haemorrhagic enteritis and turkeyrhinotracheitis. The sequence may alternatively encode paramyosin (amuscle protein common to all invertebrate parasites) or an antigenicpart thereof, somatostatin or a growth-promoting part thereof or animmune regulator.

[0044] The vectors in accordance with the invention may providemultivalent vaccine protection. For example, a vaccine comprising ILTVcarrying an MDV antigen coding sequence would be expected to protectagainst ILT and Marek's Disease.

[0045] Furthermore, the mutant ILTV viruses themselves are potentiallyuseful in vaccines as attenuated viruses, without necessarily having aheterologous sequence inserted.

[0046] A convenient process for preparing the deletional or insertionalmutants of the second aspect of the invention comprises simplyintroducing into a suitable cell, for example by co-transfection, adeletional or insertional mutant version of the appropriate region (forexample, the TK region) and either whole viral DNA or a whole virus (forexample the wild-type virus). The naked DNA of such viruses has beenfound to be infectious, provided that it has not been sheared. A calciumphosphate precipitate of the DNA is generally advantageous. Suitablecells include chicken embryo fibroblasts, chicken kidney cells and duckembryo fibroblasts, all preferably grown in sub-confluent monolayers inPetri dishes.

[0047] The transfected DNA and the whole viral DNA will then recombinewith one another in the infected cells by homologous recombination andthe desired recombinants can be screened for, for example by thedetection of hybridisation to suitable probes or by an immunoassay usingsuitable antibodies to the gene product of the region in question.

[0048] For homologous recombination to take place, the viral DNA mustreplicate. At present, no cell-free replication system for MDV, HVT orILTV is known. However, if such a system becomes available, then theprocess of the invention could be operated therein. The environment inwhich the replication and recombination occur is not critical.

[0049] The ILTV and HVT regions which were identified above as beingresponsible for encoding immunologically useful viral antigens can beinserted into suitable vectors, for example into HVT or into othervectors such as fowlpoxvirus, bacteria or fungi. In the case of viralvectors, especially herpesvirus vectors and poxvirus vectors, suchinsertion can be achieved by recombination betwen the antigen-encodingsequence, flanked by suitable non-essential sequences, and the vector'sgenome in a suitable host cell as described above. A promoter which Lsendogenous to the host will usually be used to control expression of theheterologous (viral antigen-encoding) sequence. In the case of bacteriaand fungi, the antigen-encoding sequence may be inserted using known oryet-to-be-discovered techniques of DNA manipulation. A non-pathogenicstrain of Salmonella may be used as such a host. The heterologoussequence may be inserted into the host's genome or be carried on anindependently-replicating plasmid.

[0050] The flanking sequences which are used may comprise all, virtuallyall or less of the region into which the heterologous sequence is to beinserted. If all the region is employed, then the sequence of thatregion will clearly still be present in the resulting virus, but thefunction of that region will have been deleted. If less than the wholeregion is used as flanking sequences, then the result will be astructural as well as functional deletion. Either approach may be used.

[0051] Thus, the construction of deletional or insertional mutants ofILTV can yield improved vaccines. Alternatively, the expression of ILTVglycoproteins or other ILTV proteins engineered into HVT, fowl pox orother vectors can constitute effective vaccines.

[0052] To prepare a vaccine in which HVT, MDV or ILTV is the virus orvector, the virus is grown in suitable cells such as chick embryofibroblasts in a standard culture medium which as 199 medium (Wellcomeor Flow Laboratories) for 3 to 4 days at about 37° C. The cells areharvested by scraping from the surface of the culture or bytrypsinisation and suspended in medium containing 1 mM EDTA or 10%dimethyl sulphoxide and in either case 4% calf serum before storage inliquid nitrogen in sealed ampoules.

[0053] For vaccination, typically. day-old chicks are injectedintramuscularly with about 1,000 plaque-forming units. Immunity followswithin a few days.

[0054] It should be noted that MDV and HVT are cell-associated virusesand are infectious only when present in cells. Thus, a vaccine based onsuch viruses will always include suitable infected cells.

[0055] The vaccines of the invention may be used to protect any fowlsusceptible to ILTV or HTV, including commercially-reared poultry suchas chickens, turkeys. ducks and quail.

[0056] Preferred aspects of the invention will now be described by wayof example and with reference to the accompanying drawings, in which:

[0057]FIG. 1 is a map of the MDV genome showing in part the BamH1 sitedistribution and the location of the gB and TK genes;

[0058]FIG. 2 (on 18 sheets) shows the nucleotide sequence of the gB geneof the RB1B strain of MDV, with the numbering referring to the MDVnucleotides, the sequence of part of the HVT gB gene shown under theline, homologies indicated by vertical bars, and amino acid differencesbetween MDV gB and HVT gB shown above the line;

[0059]FIG. 3 is a map of the HVT genome showing the positions of the gH(hatched), TK (solid black) and manor capsid protein (MCP, dotted)genes, with HindIII sites shown as “H”;

[0060]FIG. 4 (on 8 sheets) shows the nucleotide sequence of most of theHVT gH gene, with the corresponding amino acid sequence shown above theline;

[0061]FIG. 5 (on 10 sheets) shows the nucleotide sequence of the HVT TKgene, with the numbering referring to the HVT nucleotides. the sequenceof Dart of the MDV TK gene shown under the line, homologies indicated byvertical bars and amino acid differences between MDV TK and HVT TK shownabove the line;

[0062]FIG. 6 (on 6 sheets) shows the nucleotide sequence of the gC geneof the RBIB strain of MDV, with corresponding amino acids shown abovethe line;

[0063]FIG. 7 (on 11 sheets) shows the nucleotide and predicted aminoacid sequence of a 5400 base pair region of the ILTV genome containingthe TK gene cluster. Amino acid sequences predicted for the products ofthe major open reading frames (ORFs) are indicated in the single lettercode below the sequence for the strand and above the sequence for thecomplementary strand. The locations of potential ‘TATA’ boxes areunderlined. ORF 4 is the ILT TK gene sequence;

[0064]FIG. 8 is a representation of the gene organisation in theTK-containing part of the ILTV genome. Overlapping pUC 13 plasmid clonescontaining the EcoR1 (pILEc1) and BglII (pILBg2) generated fragments ofILTV DNA are indicated. Open reading frames (ORFs) are depicted as openboxes with the direction of transcription indicated by the arrow;

[0065]FIG. 9 shows part of the nucleotide sequence of the ILTV gB gene;

[0066]FIG. 10 shows part of the nucleotide sequence of the ILTVribonucleotide reductase (large subunit);

[0067]FIG. 11 shows part of the nucleotide sequence of the HVT homologueof the VZV62/HSV-1 IE 175 gene;

[0068]FIG. 12 shows part of the nucleotide sequence of the HVTribonucleotide reductase (large subunit) gene:

[0069]FIG. 13 (one 2 sheets) shows part of the nucleotide sequence ofthe MDV ribonucleotide reductase (large subunit) gene:

[0070]FIG. 14 shows part of the nucleotide sequence of MDV homologue ofribonucleotide reductase (small subunit) gene;

[0071]FIG. 15 shows part of the nucleotide sequence of the MDV homologueof the HSV-1 IE-175 gene;

[0072]FIG. 16 shows part of the MDV homologue of the HSV-1 IE-68 gene:

[0073]FIG. 17 is a schematic representation of homologous recombinationat a non-essential region of a viral genome and a homologous region ofDNA cloned within a plasmid vector; and

[0074]FIG. 18 is a map of plasmid pILBg2, showing restriction sites andthe locations of the TK gene and ORFs 3 and 5.

EXAMPLES General Approaches

[0075] Selected short sequences of the avian herpesviruses cloned in thebacteriophage vector M13 were used as probes to identify longerfragments that might contain the entire genes of interest. This wasachieved by Southern blot hybridization of restriction fragments. Fulldetails are given below.

[0076] Virus Strains.

[0077] The ‘highly oncogenic’ strain RB1B of MDV [Schat, K. A. et al(1982) Avian Pathol. 11, 593-605] was obtained from Professor B. Calnek,Cornell University, Ithaca, U.S.A. The virus received has been plaquepurified in chicken kidney veils in tissue culture. It was passagedtwice in SPF RIR chickens and 4 times in chick embryo fibroblasts (CEF).Its ‘highly oncogenic’ nature was demonstrated by a high incidence ofgross tumours when inoculated in genetically resistant N-line chickens.

[0078] The FC126 strain of HVT [Witter, R. L. et al (1970) Am. J. Vet.Res. 31, 525-538], obtained from the Wellcome Research Laboratories,Beckenham, Kent, had been passaged 14 times in CEF. It was subsequentlygrown in duck embryo fibroblasts (DEF) and CEF in our laboratory. It wasthen plaque-purified and grown further in CEF. Viral DNA used forcloning in the present work was extracted from virus that had beenpassed 29 times since the original isolation.

[0079] The Thorne strain of ILTV was passaged twice in eggs, once inchicken kidney cells (CKC) and plaque-purified three times in CKC.

[0080] Tissue Culture.

[0081] CEF were grown in roller bottles in 199 medium (Wellcome),supplemented with penicillin, streptomycin, Fungizone (Regd. T. M.) andcalf serum as described previously [Ross, L. J. N. et al (1975) J. gen.Virol. 28, 37-47].

[0082] CKC were grown in 10 cm Petri dishes [Churchill, A. E. and BiggsP. M., (1967) Nature, 215, 528-538].

[0083] Isolation of MDV DNA.

[0084] Cell associated RB1B was inoculated onto confluent monolayers ofCEF in roller bottles at a multiplicity of infection of approximately0.001 plaque-forming units (pfu) per cell, and the cultures wereincubated at 37° C. After 3 days, the medium was discarded and replacedwith fresh 199 medium containing 2% calf serum. Cells were harvested forvirus purification after 2 to 3 days when cytopathic effect wasextensive. Virus was obtained by rate zonal centrifugation of thecytoplasmic fraction of infected cells [Lee, Y. S. et al (1980) J. gen.Virol. 51, 245-253]. Viral DNA was extracted by treating purified viruswith sarcosyl, proteinase K and Tris buffer pH 9 overnight at 37° C. andpurified by rate zonal centrifugation in glycerol gradients as describedpreviously (Lee et al, 1980). High molecular weight viral DNA wasprecipitated with ethanol and resuspended in 10 mM Tris pH 7.5 im 1 mMEDTA (TE).

[0085] Isolation of ILTV DNA.

[0086] (a) Infected CKC were harvested 2-3 days after inoculation,washed in PBS, and resuspended in ice-cold TE by vortexing. Cells werelysed by addition of the non-ionic detergent NP40 (final 1%) vortexingand incubation on ice for 15 min. After treatment with RNAse, thepreparation was centrifuged at 2000 rpm for 5 min in a bench topcentrifuge (Centaur). The supernatant was collected and incubated at 37°C. for 30 min in the presence of SDS (final 1%) and proteinase K (final0.5 mg/ml). The mixture was extracted twice with phenol-chloroform andonce with chloroform and the DNA was then precipitated with ethanol and1/10 vol of 3M sodium acetate.

[0087] (b) Viral DNA was also isolated from the media of virallyinfected cells in the following way. The media of infected cells wereharvested at 2-3 days post infection and centrifuged at 3000 for 5 minsat 4° C. rpm in a bench centrifuge. The supernatant was collected andcentrifuged at 19K rpm in an ultracentrifuge (Sorvall) for 1 hr at 4° C.The viral pellet was resuspended in TE, digested with RNAse A, thendisrupted with SDS and proteinase K as described above. Finally, DNA wasextracted from the disrupted-virus as described above.

[0088] Cloning of MDV DNA.

[0089] One fg of MDV DNA was cut with the restriction enzyme BamH1 andligated to BamH1-cut, dephosphorylated pUC13 DNA (Pharmacia). CompetentE. coli strain TG1 cells were transformed according to standardprocedures [Hanahan, D. (1983) J. Mol. Biol. 166, 557-580] and weregrown in the presence of ampicillin and X-gal. White colonies werepicked and tested for the presence or MDV inserts by hybridization tonick-translated MDV DNA [Grunstein M. and Hogness, D. S. (1975) Proc.Natl. Acad. Sci. U.S.A. 72, 3961]. Positive colonies were cultured insmall volume and plasmid DNA isolated by the procedure of Holmes, D. S.and Quigley, M. [(1981) Anal. Biochem. 114, 193-297]. The size of theinserts was determined by electrophoresis of BamH1 digests of therecombinant DNA in agarose gels. Plasmids containing MDV inserts rangingfrom less than 1 to 18 Kbp were obtained.

[0090] Cloning of ILTV DNA.

[0091] EcoR1 and Bg1II libraries of ILTV DNA were obtained by cloningdigests of viral DNA in pUC13 as described above.

[0092] Random Sequencing of Viral DNA.

[0093] Sonicated fragments of viral DNA were cloned into SmaI-cut,dephosphorylated M13.mp10 (Amersham International PLC) and plaquescontaining MDV inserts were identified by hybridization to MDV DNA. Thesequence was determined by the dideoxy method [Sanger, F. et al (1977)Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467] using ³⁵S dATP).

[0094] The same procedure was used to sequence cloned fragments of MDV,HVT and ILTV DNA except that plaques were identified by hybridization tolabelled insert so as to avoid colonies containing pUC13 fragments.

Example 1 gB Gene of MDV

[0095] An M13 clone of HVT homologous to the gB gene of VZV and HSVhybridized to BamH1 fragment I3 of MDV (see FIG. 1). Sequencing of thisfragment obtained from a BamH1 library of the RB1B strain of MDV showedthat two thirds of the gene, starting with the NH₂ terminus, wascontained within I3. The remainder of the gene was identified in theadjacent restriction fragment K3. FIG. 1 shows the map position of thegene which is 2.6 Kbp long. Its mRNA has been estimated to beapproximately 2.8 Kb. The translated protein is 865 amino acids long(FIG. 2). This includes approximately 20 amino acids which may be partof a signal sequence domain. The primary translated sequence of MDV gBhas a few features in common with gB of other herpes viruses such as thealignment of cysteine residues and the presence of hydrophobic sequenceswhich are presumably capable of spanning a lipid bilayer [Pellet, P. E.et al (1985), J. Virol. 53, 243-253]. However, MDV gB has only 48% aminoacid similarity with gB of HSV and has many unique features such as theinsertion of 23 amino acids (residues 1851-1920, FIG. 2) and thepresence of extra sites with glycosylation potential. Comparison of thesequence of MDV gB with limited sequence data (702 bases) available forHVT gB (FIG. 2) has shown 76.9% nucleic acid similarity and 87.1% aminoacid similarity between these two glycoproteins. Amino acidsubstitutions in HVT gB compared to MDV gB were particularly marked in aregion (residues 1323-1433) equivalent to a domain of HSV gB associatedwith virus neutralization [Pellet P. E. et al (1985) as above]. Aminoacid substitutions between MDV and HVT gB were also noted in otherregions of unknown function.

Example 2 gH Gene of HVT and gH Gene of MDV

[0096] An M13 clone of HVT containing sequences homologous to HSV gH wasisolated during our earlier work on gene identification and mapping(Buckmaster et al (1988) as above). This clone, when used as a probe,hybridized to a 6 Kbp HindIII fragment of HVT (FIG. 3). Sequencingrevealed that this fragment contained approximately one quarter of thegH gene including the carboxy terminus. The adjacent HindIII fragment(3.2 Kbp) containing the remainder of the gH gene was identified byhybridization using a cloned HpaI fragment of HVT which overlapped theHindIII site. FIG. 4 shows the sequence of the coding region of the gHgene of HVT (2.3 Kbp) and flanking sequences. The % amino acid identitybetween the gH gene of HVT and its homologue in HSV1, VZV and EBV wasonly 20, 24 and 20 respectively (estimated from maximised amino acidoverlaps of 630, 644 and 153 respectively).

Example 3 TK Gene of HVT and TK Gene of MDV

[0097] The whole coding region of the TK gene of HVT (1053 bp) wascontained within the 3.2 Kbp HindIII fragment described above (FIG. 3).The sequence of the entire gene and flanking regions is shown in FIG. 5.Similarly the whole of the MDV TK gene is contained within the 3.6 KbpBamH1 K2 fragment of MDV (FIG. 1). The sequence of MDV TK genedetermined so far is shown in FIG. 5. Comparison of the MDV and HVT TKsequences indicates that the two genes have approximately 60% amino acididentity (estimated from 276 amino acid overlap). By contrast, the %amino acid identities between the TK gene of HVT and the TK genes of HSV1, VZV and EBV are only 30, 27 and 24 respectively (estimated from aminoacid overlaps of 320, 332 and 193 respectively). The predicted aminoacid sequences of HVT and MDV TK show characteristic ATP and/or CTPbinding site motifs described for a number of virus and eukaryoticproteins that are associated with phosphorylation (Gentry, G. A. (1985)Proc. Natl. Acad. Sci. U.S.A. 82, 6815-6819). These conserved sequencesare examples of useful sites for insertion and expression of foreigngenes and for producing TK⁻ deletion mutants.

Example 4 A Antigen Gene of MDV (gP57-65) (gC homologue)

[0098] The A antigen gene is of interest in vaccine development both asan immunogen (it encodes a major glycopolypeptide product) and alsobecause we have identified it as the homologue of HSV gC, a potentialnon-essential region. The A antigen gene was mapped within the BamH1 Bfragment of MDV (Isfort et al 1987), and the nucleotide sequencedetermined for the GA strain of MDV (Coussens and Velicer, AbstractOP18.51, VII International Congress of Virology, Aug. 9-14, 1987Edmonton, Canada; J. Virol. 62, 2373-2379). During the random sequencingstudies described earlier (Buckmaster et al 1988), we identified an M13clone (No. 130) which came from the A antigen gene. This clone was thenused to identify a 2.3 Kbp EcoR1/PvuII fragment from the RB1B strain ofMDV containing the A antigen. This fragment was cloned into a SmaI/EcoR1cleaved pUC13 vector by standard protocols. One plasmid (pMB419) wassequenced by the M13 dideoxynucleotide method. The sequence of the MDVRB1B A antigen and the predicted amino acid sequence of the protein arepresented in FIG. 6. The A antigen regions of MDV and HTV arenon-essential genes and they can therefore be used as sites in MDV andHVT into which other genes can be inserted into the virus by homologousrecombination. Several lines of evidence support this as outlined below.

[0099] 1) During our study we isolated and sequenced another RB1B Aantigen clone. This had one extra T residue in the string of T's 45bases 3′ to the A antigen ATG codon. This extra T would cause aframeshift which would make it impossible for the gene to encodefunctional A antigen. As it is probable that this gene was cloned from areplicating MDV, the results suggest that the A antigen is non-essentialto the virus.

[0100] 2) On conducting a similarity search it became clear that the MDVA antigen gene is the homologue of HSV gC and PRV gpIII glycoproteins.Both of these homologous genes are known to be non-essential [for theHSV homologue, see Rosenthal et al (1987) J. Virol. 61, 2438-2447].

[0101] 3) Strains of MDV lacking A antigen as judged by agar geldiffusion tests [Churchill, A. E. et al (1969) J. gen. Virol. 4,557-564] or producing low levels using the more sensitive 2Dradio-immunoprecipitation (van Zaane, D. et al (1982) Virology 121,133-146] have been reported.

[0102] Furthermore, in view of the fact that the A antigen is a majorsecreted glycoprotein, it may be a particularly suitable location forthe presentation of foreign epitopes within the A antigen as soluble,secreted proteins. This may be achieved by cloning oligonucleotidesencoding these epitopes in frame within the A antigen gene.

Strategies for Introducing Genes into HVT and ILTV Vectors

[0103] Two possibilities can be envisaged. 1) Insertion intonon-essential genes of the vector. 2) Substitution of foreign gene forcorresponding gene of the vector. This would be possible only in regionswhich already have substantial homology as may be the case between somegenes of MDV and HVT.

Example 5 Insertion Into Non-essential Genes of HVT, ILTV or MDV

[0104] (a) Insertion at the TK Locus of the Vector.

[0105] 1) HVT, ILTV or MDV may be used as vectors for insertion andexpression of avian herpesvirus genes. In particular gB, gD, gH or gC ofRB1B MDV may be inserted into ILTV. Also gB and BS-17 of ILTV may beinserted into HVT or MDV. One may use the promoter associated with theinserted gene or use heterologous promoters, including those of adifferent class of genes (for example the immediate early promoter tooptimise expression of PUB).

[0106] 2) ILTV may be used as a general vector for the insertion andexpression of genes unrelated to avian herpes viruses and likely torequire manipulation of promoters for optimal expression.

[0107] The procedure to be used for gene insertion is substantially asdescribed previously for the insertion of hepatitis antigen in HSV [Shihet al, 1984 as above].

[0108] MDV and HVT DNA obtained as described above is infectiousprovided that precautions are taken not to shear the DNA duringextraction. Calcium phosphate precipitates of viral DNA prepared asdescribed by Stow and Wilkie [(1976) J. gen. Virol. 33, 477] were addedto sub-confluent monolayers of CEF. After absorption for 1 h at 37° C.,culture medium was added and cultures were incubated for 1 or 2 daysuntil confluent. Monolayers were then trypsinised, replated (1:1 or 1:2)in 199 medium (Wellcome) containing 2 to 4% calf serum and incubated at37° C. until plaques developed, usually after 4 to 5 days. Approximately200 plaques may be obtained per μg of HVT DNA and approximately 50 perμg of MDV DNA.

[0109] Restriction enzyme sites than could be used for the insertion offoreign antigens into the TK of HVT strain Fc-126 include: BanII,Bsp1286, DraIII, EcoRI, HincII, HpaI, NheI and NspbII.

[0110] Some of these enzymes also have sites in the plasmid vector intowhich the virus DNA fragments are cloned. Thus in order to linearize theclone DNA without also cutting within the vector, partial digests may becarried out.

[0111] None of the above enzymes should cause any disruption to flankinggene, HSV-1 homologues of which are known to play an important role invirus multiplication.

[0112] For homologous recombination and isolation of recombinant virus,genes of interest are inserted within non-essential genes such as TK orgC and co-transfected with wild-type viral DNA at molar ratios rangingfrom 10:1 to 2:1 as described above. Alternatively, intact wild-typevirus may be used for co-infection.

[0113] Virus recombination may be detected by ‘plaque lifts’ whichinvolve transfer of infected cells and released virus which have adheredto the agar overlay to nitrocellulose and hybridization of the denaturedDNA released from the cells and virus to suitable probes as described byVillareal, L. et al (1977) Science 196, 183-185. Virus which hybridizesto. the probe may be recovered from the monolayer.

[0114] A similar procedure may be used to isolate recombinant viruswhich expressed epitopes of interest. In this instance thenitrocellulose “plaque lifts” are treated with antibody and the presenceof bound antibody revealed using a suitable detection system such aslabelled protein A or phosphatase conjugated anti-globulin antibody.

[0115] The gene of interest with appropriate promoters is first insertedwithin the cloned TK gene (FIG. 7). The recombinant DNA is thenco-transfected with infectious DNA of the vector in chick embryofibroblasts or chicken kidney cells and TK⁻virus may be selected bygrowth in medium containing acyclovir [Ross, N. (1985) as above] or FMAU[Schat, K. A. et al (1984) Antiviral Research 4, 159-270].Alternatively, or in addition, plaques are screened for the presence ofthe gene of interest using ‘plaque lifts’ on nitrocellulose andhybridization to any relevant labelled probe. Plaques are also screenedfor expression of the epitopes of interest using monoclonal antibodiesor antipeptide antibodies.

[0116] The main advantage of this strategy is that the selectionprocedure increases the chances of obtaining virus recombinantscontaining the gene of interest. It also offers the opportunity of usingdifferent promoters for optimum expression. Thus the use of an immediateearly promoter may allow expression in latently infected cells.

[0117] (b) Insertion at the gC Locus of the Vector.

[0118] Since the A antigen (HVT and MDV homologues of HSV gC) is notessential for virus growth in vivo and in vitro (see section on gCabove) it is a potentially useful site for the insertion and expressionof foreign genes. Moreover, since it is one of the most abundantantigens and is excreted, it may be particularly useful for enhancingthe immunogenic properties of foreign proteins. The isolation of virusrecombinants at this locus may be achieved by first inserting at leastpart of the gene of interest in frame within the gC gene and thenco-transfecting with infectious viral DNA. Screening of virus plaqueswith sequence specific probes or with specific antibody allows theisolation of recombinants.

Example 6 Substitution of ILTV Genes for their Homologues in HVT

[0119] Substitution may be achieved by co-transfection of cloned ILTVsequences and infectious HVT DNA as described in Example 5. Substitutionof genes derived from ILTV For their counterparts in HVT may beeffected.

[0120] Recombinants expressing ILTV sequences and epitopes may bedetected using ILTV-specific monoclonal antibodies or anti-peptideantibodies raised against unique ILTV sequences as described above.

[0121] The advantage of this procedure is that it is relatively simpleand does not require manipulation of promoters. However, it may belimited to genes which share substantial homology.

Example 7 Strategies for obtaining TK⁻Mutants of ILTV

[0122] Deletion Mutants.

[0123] Deletions may be introduced within any suitable part of the gene,for example the domains of the gene that are required for its functionas a phosphorylating enzyme such as ATP and CTP binding sites. This maybe achieved by restriction enzyme digestion, for example with SnaB1 orBclI, and religation of appropriate fragments followed byco-transfection with infectious viral DNA or transfection intovirally-infected cells. Reference may be made to FIGS. 7 and 8, and tothe map of plasmid pILBg2 (FIG. 18), in choosing restriction enzymes andso on. TK⁻ virus may be selected in the presence of acyclovir [Ross, N.(1985) as above] or FMAU [Schat, K. A. et al (1984) as above].Plaque-purified clones may then be tested for the absence of the deletedportion of the TK gene by hybridization.

[0124] The deletion mutants of ILTV may be used themselves as attenuatedviruses for vaccine preparation, or may have sequences for heterologousantigens inserted.

[0125] Insertional Mutants.

[0126] A functional β-galactosidase gene under the control of aherpesvirus promoter or any other suitable sequence or a single base isfirst introduced in a domain of the TK gene which is essential for TKactivity. The recombinant DNA is then co-transfected with infectiousviral DNA or transfected into virally-infected cells to allow homologousrecombination to occur. Selection in the presence of acylovir or FMAUwill yield TK⁻ insertional mutants. If a β-galactosidase gene isintroduced, mutants can be detected by the production of blue plaques inthe presence of X-gal.

[0127] The TK gene and surrounding sequences may be subcloned intoanother suitable vector if necessary.

Example 8 Insertion of MDV RB1gB Gene into HVT

[0128] (Not within the scope of the invention, but illustrates ananalogous technique).

[0129] The HVT TK gene is cloned in the plasmid vector pUC13 to generatea plasmid, which may be termed pTK1B. This plasmid is linearised with,for example, the restriction endonuclease Rsr II which cleaves theplasmid only within the TK gene (nucleotide position 197 in FIG. 5,enzyme recognition sequence CGGACCG). The “sticky” ends thus generatedare end repaired by standard techniques (see “Molecular Cloning: aLaboratory Manual”, ed. Maniatis T., Fritsch E. F., and Sambrook J. ColdSpring Harbor Laboratory 1982).

[0130] The RB1B gB was originally cloned on two plasmids which weretermed RB1B-BamH1-I₃ and RB1B-BamH1-K₃. (Note I₃ had lost one BamH1 siteduring cloning.) To generate a complete gB copy on one plasmid, bothplasmids were cleaved with BamH1 and the fragments ligated. However, thecomplete gB gene was later obtained independently on an EcoRI/SalIfragment. Ross et al, J. gen. Virol (1989) 70, 1789-1804 providesfurther information regarding the manipulation of viral genes.Recombinants containing the desired configuration can be identified byrestriction enzyme analysis of plasmid DNA's.

[0131] The recombinant plasmid is then cleaved with EcoR1, the ends arerepaired and the plasmid is cloned into PTK1B prepared as above. Therecombinant plasmid is then introduced into cells containing HVT virus(viral DNA) and homologous recombination will introduce the gB gene intothe TK gene. HVT viral recombinants can be selected with acyclovir orFMAU or alternatively detected with labelled gB probes.

Example 9 RB1B gC (A Antigen) Gene into HVT

[0132] Blunt ended PTK1B is prepared as in Example 8. The RB1B gC iscleaved from the plasmid pMB419 (Example 4) with the restrictionendonucleases EcoR1and HindIII (site within the pUC13 polylinker). Thesticky ends generated are again end-repaired by standard protocols. Theend-repaired gC fragment is then cloned into the linearized end-repairedpTK1B as in Example 8. (The cloning can be verified by analysis of theresulting clones with restriction enzymes, probing with radio-activelylabelled fragments, or DNA sequencing, or any combination of these).

[0133] The resulting plasmid with the RB1B gC gene cloned into the HVTTK gene can then be introduced into the HVT genome by transfecting theplasmid into HVT-infected cells using calcium phosphate precipitation orelectroporation. Homologous recombination, involving cross-overs eitherside of the gC gene, between the HVT virus and the flanking sequences ofthe HVT TK plasmid will carry the RB1B gC gene into the HVT viralgenome. Viral recombinants can be selected for (as they are TK⁻) oridentified (eg by probing) as described above.

[0134] In analogous ways, the sequence information given above and inthe Figures can be used to design cloning strategies for the insertionof these genes and others into the non-essential genes of the ILTVdescribed here or to generate combinations of antigen genes into ILTV.

What is claimed is:
 1. A recombinant ILTV synthetically modified by amutation in an ILTV gene such that the recombinant ILTV is attenuated asa result of said mutation.
 2. The ILTV according to claim 1 , whereinthe mutation is in the TK, ORF3 or ribonucleotide reductase, largesubunit, gene.
 3. The ILTV according to claim 1 , synthetically modifiedby the presence, in a non-essential region of the ILTV genome, of DNAnot naturally occurring in ILTV.